Methods and compositions for stabilizing dried biological materials

ABSTRACT

The present invention relates to methods for producing dried formulations of biopharmaceutical agents that aim to minimize the loss of activity of the agents upon drying and to provide dried formulations with an extended shelf life. The method comprises the step of drying an aqueous solution comprising, in addition to the biopharmaceutical agent, at least an amino acid, a polyol and a metal salt. Preferably the amino acid is glutamate, the polyol is sorbitol and optionally also mannitol and the metal salt is a magnesium salt. The solution is dried by vacuum drying or by lyophilization. The methods are particularly useful for preparing dried formulations of viruses such as poliovirus or respiratory syncytial virus to be used for vaccination. The invention also relates to dried formulations prepared in accordance with the methods of the invention and to their use as medicaments, e.g. as vaccines.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage of International ApplicationNo. PCT/NL2013/050139 filed on Mar. 5, 2013, published as WO2013/133702, which claims priority to European Application No.12158086.4 and U.S. Provisional Application No. 61/606,577, both filedMar. 5, 2012. The contents of these applications are herein incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates the methods and compositions forstabilizing and preserving dried biological materials. These biologicalmaterials include lyophilized or vacuum-dried preparations ofbiopharmaceuticals such as vaccines, in particular viral vaccines. Theinvention therefore relates to the field of production and formulationof biopharmaceuticals and to the field of vaccinology.

BACKGROUND OF THE INVENTION

The long-term storage of biological compounds poses a unique challenge,considering that these compounds are usually fragile and vulnerable.Very few biological compounds are sufficiently stable in liquidenvironment, in solution or suspension, to allow them to be isolated,purified and stored at unrefrigerated conditions, especially at roomtemperature or higher temperatures as a solution for anything more thana short period of time.

One way to improve the stability of biopharmaceuticals is by convertingthem into a dry state [17]. Both commercially and practically, storageof biological compounds in dry form carries with it enormous benefits.Successfully dried reagents, materials and tissues have reduced weightand require reduced space for storage not withstanding their increasedshelf life. This is not only of use for the final product, like in finallots of vaccines for use within 3 months to 2 year, but also forstockpiling (1-50 year or more years of storage) seedlots, bulks orfinal lots. Room temperature storage of dried materials is moreover costeffective when compared to low temperature storage options and theconcomitant cost. In addition, several routes of delivery, includingpulmonary delivery of powders, dermal delivery by coated or dissolvingmicroneedles, parenteral delivery by powders or dissolvable needlesdepend on sophisticated ways of formulations. There exist severaltechnologies for producing dried biological compounds, including spraydrying, vacuum drying, air-drying, coating, foam-drying. One of theoldest and commonly used technique is freeze-drying, also calledlyophilization. For a long period of time freeze-drying was seen as moreof an art than a science, which hindered a scientific approach andresearch.

The most commonly used method for preparing solid biopharmaceuticals islyophilization. This process consists of a freezing step followed by twodrying steps, the primary drying where frozen water is removed bysublimation and the secondary drying where the non-frozen ‘bound’ wateris removed. Either freezing or drying stresses can modify thethermodynamic stability of biopharmaceuticals and can induce orfacilitate protein unfolding. Unfolding can lead to irreversibledenaturation of the biopharmaceutical, but may also reduce the storagestability in the dry state [19]. For a stable lyophilisate, excipientsserving as stabilizer and/or bulking agents are used. Differentcompounds, such as sugars, polymers, amino acids and surfactants, havebeen shown to improve the stability of biopharmaceuticals duringlyophilization and subsequent storage [18, 20]. In literature severalmechanisms are described how excipients are believed to protectbiopharmaceuticals like proteins and vaccines, during freezing, dryingand subsequent storage. Understanding the cryo- and lyoprotectionmechanisms of different stabilizers is important in the development of arational formulation and process design for a stable lyophilized vaccine[21].

During freezing, the physical environment of a biopharmaceutical changesdramatically leading to the development of stresses that impact theintegrity of the proteinaceous biopharmaceutical. The most criticalstresses to which a biopharmaceutical is exposed during freezing are lowtemperature, freeze-concentration and the formation of ice [18, 19, 21,22]. Cold denaturation is the phenomenon whereby biopharmaceuticals losetheir compact folded structure as a result of a temperature drop. Thecurrently accepted explanation for cold denaturation is based on achange in the contact free energy between water and non-polar groups atcolder temperatures, which would weaken the hydrophobic interaction andthus disrupt biopharmaceutical structure [19, 20, 23]. Due to iceformation, the concentration of all solutes increases dramaticallyduring freezing. All changes related to concentration, such as ionicstrength, crystallization of solutes and phase separation, maypotentially destabilize a biopharmaceutical [21].

The initial relative composition and pH of the formulation cancircumvent detoriation of the biopharmaceutical by freezing stresses.For example, it has been found with many biopharmaceuticals thatincreasing the biopharmaceutical concentration in the formulationrelatively to other excipients before freezing will increase thestability of the biopharmaceutical during freeze-thawing [24].Similarly, an initial pH that is optimal for the biopharmaceutical insolution will give the highest recovery of intact biopharmaceuticalafter freeze-thawing [20].

Even after optimization of all these factors, many biopharmaceuticalsstill denaturize during freeze-thawing, therefore additives are neededto minimize protein/biopharmaceutical denaturation. Different excipientsthat come from very dissimilar chemical classes are able to givecryoprotection. According to the ‘solute exclusion hypothesis’,cryoprotectants have been shown to preferentially not to be in contactwith the surface of biopharmaceuticals in aqueous solutions [24]. Thethermodynamic phenomenon of solute exclusion in the presence of variousbiopharmaceuticals has been determined for various excipients, such assalts, amino acids, methylamines, polyethylene glycols, polyols,surfactants and sugars [19, 21, 24, 25].

The ‘vitrification hypothesis’ is a widely known kinetic mechanism.According to this mechanism, both freeze-concentration and a temperaturedrop increase viscosity, reduce mobility and slow all dynamic processes.When the system reaches a glassy state, all molecules in the glassbecome physically (e.g. denaturation, aggregation) and chemically (e.g.oxidation, hydrolysis, deamidation) immobile and the rate constant ofbiopharmaceutical degradation is reduced [19, 26].

Ice-water interfaces formed during freezing may cause surfacedenaturation. Addition of surfactants may drop surface tension of thebiopharmaceutical solution and thus reduce biopharmaceutical adsorptionand aggregation [25, 27].

Polymers could stabilize biopharmaceuticals by raising the glasstransition temperature of the formulation and by inhibitingcrystallization of small stabilizing additives, like disaccharides [18,22]. Amino acids may protect biopharmaceuticals as well from freezingdenaturation by reducing the rate and extend of buffer saltcrystallization and thus suppressing the pH shift [18].

In an aqueous solution biopharmaceuticals are fully hydrated, whichmeans that the biopharmaceutical has a monolayer of water covering andinteracting (by hydrogen bonds) with the biopharmaceutical surface [28].Drying removes part of the hydration shell and this may disrupt thenative state of the biopharmaceutical leading to denaturation. In orderto prevent denaturation during drying protectants are required. Animportant stabilization mechanism of such protectants is called the‘water substitution hypothesis’ [18-20, 29]. Sugars, such as sucrose andtrehalose, polyols [20, 30] and amino acids [31] are able to formhydrogen bonds with the dried biopharmaceutical. As such they can act asa water substitute, when the hydration shell is removed. The formationof an amorphous glass, explained above as the ‘vitrificationhypothesis’, is also a major protection mechanism.

In addition to water substitution and glass formation, many excipients,especially polymers can stabilize biopharmaceuticals by increasing theglass transition temperature (T_(g)), which is defined as the transitiontemperature between the rubbery (liquid-like) and glassy (solid-like)states. Generally, the higher the T_(g), the lower the molecularmobility in the glass (e.g. movement of the biopharmaceutical,stabilizing compounds, oxygen and water) and the more stable thebiopharmaceutical formulation during drying and subsequent storage [18,22]. Another mechanism involved in the stabilization ofbiopharmaceuticals during drying and that is applicable forpolysaccharides and other polymers, is the inhibition of crystallizationduring solute concentration of small excipients that stabilize thebiopharmaceutical during drying.

Due to their preference for the bulk environment instead of thebiopharmaceutical surface (previously named as ‘solute exclusion’), someexcipients are able to act as bulking agent, which means that theyprovide mechanical support to the final cake, improve product elegance,and prevent product collapse during drying. Mannitol and glycine arefrequently used bulking agents, because of their non-toxicity, highsolubility, and high eutectic temperature [18, 25, 32]. Most amino acidsare potential bulking agents as well as they easily crystallize [33].

If the biopharmaceutical is stable during the drying process, thenlong-term storage in the dried state is often feasible. Although ingeneral the drying process itself is most detrimental to thebiopharmaceutical, dried biopharmaceuticals may still loss theirstructure or potency during storage if not properly formulated.Aggregation is a major physical instability for biopharmaceuticalsduring storage. Different chemical degradations, like deamidation,oxidation and hydrolysis, may occur as well during storage, but thesealterations may not necessarily affect the activity ofbiopharmaceutical, depending on the location of the transformedresidue(s). Reducing sugars such as glucose and sucrose can react withlysine and arginine residues in biopharmaceuticals to form carbohydrateadduct via the Maillard reaction, a browning reaction, which can lead toa significant loss of activity of the lyophilized biopharmaceuticalduring storage.

Storage temperature is one of the most important factors affecting thestability of biopharmaceuticals in the solid state. Other factors thataffect long-term storage stability are the glass transition temperature(T_(g)) of the formulation, formulation pH and the residual moisturecontent after drying. The moisture content of a dried formulation maychange significantly during storage due to several factors, includingstopper moisture release, crystallization of an amorphous excipients ormoisture release from an excipient hydrate. Excipients that are used tostabilize biopharmaceuticals during drying may destabilizebiopharmaceuticals in the solid state if their quantities are notappropriately used in the formulation. Also the risk of crystallizationof amorphous excipients exist during storage, because the crystallinestate is thermodynamically more stable than the glassy state [39]. Manysugars and polyols have the tendency to crystallize, but this isstrongly affected by their relative amount in the formulation, storagetemperature and relative humidity [18, 40]. In addition, the relativecomposition of several excipients and the presence of non-crystallizingstabilizers, such as polymers, may inhibit crystallization ofexcipients.

Stabilization mechanisms for biopharmaceuticals in the dry state duringlong-term storage are similar to those for lyoprotection, includingformation of an amorphous glassy state, water substitution, and hydrogenbonding between excipients and the biopharmaceutical [41]. A combinationof these mechanisms is required for maximum biopharmaceuticalstabilization in the solid state [18, 42]. The final quality of alyophilized product is determined by the choice of excipients, includingbuffering, bulking and stabilizing agents, and the lyophilizationprocess.

Poliomyelitis is a highly infectious disease which mainly affects youngchildren. The disease, caused by any one of three serotypes ofpoliovirus (type 1, type 2 or type 3) has no specific treatment, but canbe prevented through vaccination. Currently, the oral poliomyelitisvaccine (Sabin OPV) is the vaccine of choice to strive for globaleradication of poliomyelitis. However, a major concern is the ability ofOPV to revert to a form that can cause paralysis, so-called vaccineassociated paralytic poliomyelitis (VAPP). Another risk of permanent useof live attenuated poliovirus is the reversion to vaccine-derivedpolioviruses (VDPV) [1]. In Western countries the use of an inactivatedSalk polio vaccine (IPV) is the current preferred way to eliminate therisk of VAPP and circulating VDPV. IPV is thought to be most suitablefor continuation of the global eradication program [1-3].

To achieve global polio eradication an (improved) IPV must beefficacious, inexpensive, safe to manufacture, and easy to administer[4]. The feasibility of current IPV in developing countries is limited,because IPV is more expensive than OPV and is administered throughinjections only [1, 3, 5]. In order to limit the expenses of IPV, WHOand RIVM are developing a non-commercial IPV for technology transfer todeveloping countries. Because the containment of the wild-type Salkpolio virus during production might be an issue, especially indeveloping countries, the new vaccine will be based on the OPV strain,Sabin (sIPV), for which the production costs is also expected to be lessexpensive For further reduction in costs, RIVM is developing sIPVformulations that show dose sparing by using an adjuvant and/or otherimmunization routes [6].

Since alternative delivery methods and improved vaccine formulationshave the potential to make vaccine delivery easier and safer [7, 8],currently several alternative vaccine delivery methods are beingdeveloped.

It appears that there are differences in heat stability between thevarious inactivated polio serotypes, with type 1 being the mostvulnerable. In the absence of any preservative type 1 deterioratesslowly after storage for two years at 4° C., while type 2 and 3 remainpotent for many years. The D-antigen content drops significantly after20 days at 24° C. and is undetectable after exposure to 32° C. for thesame period. In contrast, no significant changes in D-antigenicity wereobserved for type 2 at either of these temperatures. Type 3 remainsstable for 20 days at 24° C., but the D-antigen content dropssignificantly at 32° C. [9].

All three serotypes of IPV show satisfactory maintenance of potency whenincorporated into combined vaccines and stored at 4° C. for periodsranging from one year to over four years, based on observations made onDT-polio vaccine, which is preserved with 2-phenoxy-ethanol and adsorbedto aluminium hydroxide [9, 10]. Longer storage resulted in a decline inantigenicity, especially for type 1 [9]. The IPV as stand-alone vaccineis stable for 4 years at 4° C. and one month at 25° C. [10]. At 37° C.there is a significant loss of potency of type 1 after 1-2 days and oftypes 2 and 3 after two weeks [11, 12]. Also freezing has a negativeeffect on the potency of IPV, which is related to loss of the D-antigenstructure [12].

After polio eradication a stockpile of polio vaccines is required toanticipate on the potential risk of new polio outbreaks caused bycirculating VDPV (even after OPV cessation) [13-15] or bioterrorismattacks. In order to achieve an optimal vaccine stockpile various issuesneed to be considered. The shelf-life is an important detail, because adelayed expiration time will reduce the stockpile costs [16]. Toguarantee the potency of vaccines for many years the shelf-life ofvaccines such as IPV needs to be extended. There is thus a need forimproved formulations that extend shelf-life of biopharmaceuticals andimproved method for producing such formulations, preferably in dry form.

SUMMARY OF THE INVENTION

In a first aspect the invention relates to a method for producing adried formulation of a biopharmaceutical agent, wherein the methodcomprises drying a solution comprising a biopharmaceutical agent, anamino acid, a polyol, a metal salt and water.

Preferably, in the method of the invention, the solution consistsessentially of 1 pg-10 g per ml of the biopharmaceutical agent, 0.01-20%(w/v) of the amino acid, 0.5-20% (w/v) of the polyol, 0.005-10% (w/v) ofthe metal salt and water. More preferably, the amino acid is glutamate,arginine, histidine, asparagine, glycine or a mixture thereof; thepolyol is sorbitol, mannitol, mannose, maltitol or a mixture thereof;and the metal salt is a salt of Mg⁺⁺, Ca⁺⁺ or Li⁺ or a mixture thereof,of which Cl⁻ or SO₄ ²⁻ salts or mixtures thereof are preferred.

In the method of the invention the glutamate is preferably dissolved inthe solution in the form of monosodium glutamate.

In a preferred embodiment of the method of the invention, the solutionconsists essentially of 1 pg-10 g per ml of the biopharmaceutical agent,5-20% (w/v) sorbitol, 5-20% (w/v) monosodium glutamate, 2-10% (w/v) of amagnesium salt, preferably MgCl₂ and/or MgSO₄, and optionally 5-20%(w/v) mannitol.

In the methods of the invention, the solution preferably comprises apharmaceutically acceptable buffer and is buffered at a neutral pH. Itis also preferred that the solution is dried by vacuum drying or bylyophilization.

In the methods of the invention, the biopharmaceutical agent preferablyis an agent comprising proteinaceous material. More preferably thebiopharmaceutical agent is a virus, preferably an Enterovirus or aPneumovirus. Most preferably, the biopharmaceutical agent comprisespoliovirus of serotypes 1, 2 and 3, preferable inactivated poliovirus ofserotypes 1, 2 and 3, or wherein the biopharmaceutical agent is a humanor bovine RSV.

In a preferred embodiment of the method of the invention, the driedformulation, upon reconstitution after drying, retains at least 50% ofthe activity of the biopharmaceutical agent present in the solutionprior to drying. In another embodiment of the method of the invention,wherein the solution to be dried comprises more than one differentbiopharmaceutical agents and wherein the difference in loss ofactivities for the different agents preferably is less than 50%, wherebythe retained activity of the agent with the most loss in activity isexpressed as percent of the retained activity of the agent with theleast loss, which is set at 100%.

In a preferred embodiment of the method of the invention, the driedformulation upon reconstitution after storage for at least one week at45° C., retains at least 50% of the activity of the biopharmaceuticalagent present in the solution prior to drying. In another embodiment ofthe method of the invention, wherein the dried formulation comprisesmore than one different biopharmaceutical agents and wherein thedifference in loss of activities for the different agents preferably isless than 50%, whereby the retained activity of the agent with the mostloss in activity is expressed as percent of the retained activity of theagent with the least loss, which is set at 100%.

In a second aspect, the invention pertains to a dried formulation of abiopharmaceutical agent obtainable in any the method according to theinvention.

In a third aspect, the invention pertains to the formulation accordingto the invention for use as a medicament, preferably for inducing animmune response (in an individual) against an infectious disease or atumour.

DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to a method for producing adried formulation of a biopharmaceutical agent. The methods of theinvention are aimed at achieving at least two improvements: 1)minimizing the loss of activity of the biopharmaceutical agent upondrying of the formulation (and preferably subsequent reconstitution ofthe dried formulation); and, 2) increasing the stability, i.e.shelf-life of the dried formulation of the biopharmaceutical agent, i.e.minimizing the loss of activity of the biopharmaceutical agent uponstorage of the dried formulation. The method preferably comprises thestep comprises drying a aqueous solution comprising a biopharmaceuticalagent and one or more of an amino acid, a polyol and a metal salt.

In the method of the invention, the solution to be dried comprises,consists essentially of, or consists of: a) the biopharmaceutical agentas specified herein below and in a concentration specified herein below;b) the amino acid as specified herein below and in a concentrationspecified herein below; c) the polyol as specified herein below and in aconcentration specified herein below; d) the metal salt as specifiedherein below and in a concentration specified herein below; e) water;optionally, f) a buffer as specified herein below, in a concentrationspecified herein below and at a pH specified herein below; and,optionally, g) further ingredients as specified herein below and in aconcentration specified herein below.

The Biopharmaceutical Agent

A “biopharmaceutical agent” is herein understood to refer to abiological agent, which is physiologically active when applied to amammal, especially when applied to a human patient, preferably in apharmaceutically acceptable form. The biological agent preferably is anagent that is produced by or obtainable from a cell, although syntheticcopies of agents obtainable from a cell and chemically modified agentsobtainable from a cell are included in the term biological agent. Thebiological agent can be a protein-based agent, i.e. an agent comprisingproteinaceous material such as proteins, polypeptides and peptides. Thebiological agent may further comprises or consist of nucleic acid, e.g.DNA, RNA or a nucleic acid analogue.

A preferred biopharmaceutical agent is a virus or a virion, preferably avirus that infects mammals, preferably a virus that infects humans. Thevirus can be an enveloped virus but preferably is a non-enveloped virus.It is understood herein the term ‘virus’ as used herein include wildtype viruses as they occur in nature (e.g. natural isolates), as well as‘man-made’ attenuated, mutant and defective viruses. The term ‘virus’also includes recombinant viruses, i.e. viruses constructed usingrecombinant DNA technology, such as defective viruses, e.g. lacking(parts of) one or more viral genes and gene therapy vectors wherein partof the viral genome is replaced with one or more gene(s) of interest. Apreferred virus is Picornavirus, more preferably an Enterovirus, such aspoliovirus, Coxsackievirus, echovirus and rhinovirus. Most preferablythe virus is poliovirus. The poliovirus can be an attenuated poliovirusbut preferably is inactivated poliovirus (IPV). The poliovirus can alsobe an inactivated attenuated poliovirus The biopharmaceutical agent cancomprise one or more of the polio viral serotypes 1, 2 and 3 butpreferably the agent comprise all three polio viral serotypes 1, 2 and3. Suitable strains of serotype 1 poliovirus include but are not limitedto one or more of the Sabin 1, Mahoney, Brunhilde, CHAT and Cox strains.Suitable strains of serotype 2 poliovirus include but are not limited toone or more of the Sabin 2, MEF-1 and Lansing strains. Suitable strainsof serotype 3 poliovirus include but are not limited to one or more ofthe Sabin 3, Saukett H and G, and Leon strains. In a preferredembodiments the biopharmaceutical agent is a trivalent inactivated poliovaccine such as e.g. the Salk-IPV, containing the inactivated polioviral Mahoney strain for type 1, the inactivated polio viral MEF-1strain for type 2 and the inactivated polio viral Saukett strain fortype 3, or sIPV, containing the inactivated polio viral Sabin-1, -2 and-3 strains. Methods for inactivating polio viral strains for safe use invaccines are well known in the art and include e.g. inactivation usingformalin or beta-propiolactone (see e.g. Jonges et al., 2010, J. Clin.Microbiol. 48:928-940).

In another embodiment biopharmaceutical agent is a virus or a virion ofa pneumovirus. The pneumovirus preferably is a Respiratory SyncytialVirus (RSV), more preferably a human or bovine RSV. The human RSV mayeither be a subgroup A or B virus, and preferably is a clinical isolate,more preferably an isolate that has not been extensively passaged invitro (preferably passaged less than 10, 8, 6 or 5 times). Preferablythe (human or bovine) RSV virus is a virus comprising a viral genomehaving a deleted or inactivated G attachment protein gene, e.g. having amutation in its viral genome whereby the viral genome does not encode afunctional G attachment protein, such as e.g. the RSV ΔG and RSV ΔG+Gvirions as described in WO 2005/061698 and in Widjojoatmodjo et al.(2010, Virol. J., 7:114).

The biopharmaceutical agent is preferably present in the solution to bedried in an amount ranging between 1×10⁰ and 1×10²⁵ live and/or dead orinactivated particles per ml. The number of live particles may bedetermined by e.g. plaque forming units, cell culture or tissue culture50% infectious dose (CCID₅₀ or TCID₅₀) and other suitable virologicalassays for determining the titer of the agent. The number of dead orinactivated particles may be determined using an assay that quantifiesthe amount of antigen, such e.g. protein assays, or assays thatdetermine haemagglutination units or polio D-antigen units. Preferablythe biopharmaceutical agent is present in the solution in an amount ofat least 1×10¹, 1×10², 1×10³, 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹ or 1×10¹⁰ live and/or dead or inactivated particles per ml and/orin an amount of up to 1×10²⁴, 1×10²³, 1×10²², 1×10²¹, 1×10²⁰, 1×10¹⁹,1×10¹⁸, 1×10¹⁷, 1×10¹⁶, 1×10¹⁵ or 1×10¹⁴ live and/or dead or inactivatedparticles per ml.

The amount of the biopharmaceutical agent in the solution to be driedcan also be expressed as weight of the biopharmaceutical agent per ml ofthe solution. Preferably the biopharmaceutical agent is present in thesolution in a weight/ml ranging between 1 pg/ml and 10 g/ml. Morepreferably, the biopharmaceutical agent is present in the solution in anamount of at least 10⁻¹¹, 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴,g/ml and/or in an amount of up to 10⁻³, 10⁻², 10⁻¹, or 10⁰ g/ml. Theweight of the biopharmaceutical agent in the solution may be determinedby means known in the art per se, including e.g. protein assays. Theaforementioned weights of the biopharmaceutical agent may thus also beexpressed as grams protein per ml to be determined in a suitable proteinassay (e.g. the Bradford assay; Zor and Selinger, 1996, Anal. Biochem.236: 302-308).

In a preferred embodiment wherein the biopharmaceutical agent ispoliovirus, the amount of poliovirus in the solution preferably is atleast 0.01, 0.1, 1.0, or 10 DU/ml and up to 10.000, 1.000 or 100 DU/ml,wherein 1 DU is defined and determined with a QC-ELISA as described byWestdijk et al. [6]. In an embodiment wherein the biopharmaceuticalagent comprises a multivalent poliovirus (vaccine), it is understoodthat each polio viral serotype is present in an amount of at least 0.01,0.1, 1.0, or 10 DU/ml and up to 10.000, 1.000 or 100 DU/ml.

In a preferred embodiment wherein the biopharmaceutical agent is RSV,the amount of RSV in the solution preferably is at least 1×10¹, 1×10²,1×10³, 1×10³, 1×10⁴ TCID₅₀/ml and up to 1×10²⁵, 1×10²⁹, 1×10¹⁵, 1×10¹²,1×10¹⁰ or 1×10⁹ TCID₅₀/ml, wherein the TCID₅₀ for RSV is defined anddetermined as described by Widjojoatmodjo et al. (2010, Virol. J.,7:114).

The Amino Acid

The amino acid in the solution to be dried can be any D- or L-amino acidthat is pharmaceutically acceptable. Such amino acids include the 20standard ‘proteinogenic’ or ‘natural’ amino acids (histidine, alanine,isoleucine, arginine, leucine, asparagine, lysine, aspartic acid,methionine, cysteine, phenylalanine, glutamic acid, threonine,glutamine, tryptophan, glycine, valine, proline, tyrosine and serine),as well as non-natural amino acid such as e.g. ornithine, citrulline,selenocysteine, taurine and pyrrolysine. Preferably the amino acid inthe solution to be dried is one or more of glutamate, glutamine,arginine, histidine, asparagine, lysine, leucine and glycine, morepreferably one or more of glutamate, arginine and histidine. Thesolution to be dried may thus comprise a mixture of amino acids. Theamino acid(s) in the solution to be dried may be either optical D- orL-isomers or mixtures thereof, although preferably the amino acid(s) areL-isomers.

In a preferred embodiment, the amino acid in the solution to be dried isone or more of L-glutamate, L-arginine and L-histidine. L-glutamate mayalso be in the form of Na-glutamate, peptone, non-animal peptone,vegetable peptone. L-arginine may also be in the form ofpoly-L-arginine, peptone, non-animal peptone, vegetable peptone.L-histidine may also be in the form of Na-histidine. L-lysine may alsobe in the form of poly-L-lysine.

In a particularly preferred embodiment, the amino acid in the solutionto be dried comprises glutamate. The glutamate can be one or more ofsodium glutamate, potassium glutamate, ammonium glutamate, calciumdiglutamate, magnesium diglutamate and glutamic acid. More preferably,the glutamate is dissolved in the solution in the form of monosodiumglutamate (MSG).

The amino acid(s) preferably are present in the solution to be dried ina concentration in the range of 0.01-20% (w/v), i.e. percent weight pervolume of the solution. Thus, preferably, the amino acid(s) are presentin the solution to be dried in a concentration of at least or more than0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 8.0, 9.0 or 9.5% (w/v)and/or the amino acid(s) are present in the solution to be dried in aconcentration of no more or less than 20.0, 17.5, 15.0, 12.5, 11.0 or10.5% (w/v). Most preferably the amino acid(s) are present in thesolution to be dried in a concentration of about 10% (w/v).

The Polyol

The polyol in the solution to be dried preferably is a sugar or sugaralcohol. Preferred sugars include sucrose, trehalose, mannose anddextran. Preferred sugar alcohols include sorbitol, mannitol andmatltitol. Preferably the solution to be dried comprises at least one ormore of sorbitol, mannose, mannitol and matltitol, more preferably, atleast one or more of sorbitol, mannose, and mannitol, still morepreferably at least one or both of sorbitol and mannitol. Mostpreferably the solution to be dried comprises as polyol at leastsorbitol, optionally in combination with mannitol.

In one embodiment the polyol(s) are preferably present in the solutionto be dried in a concentration in the range of 0.1-20% (w/v), i.e.percent weight per volume of the solution. Thus, preferably, thepolyol(s) are present in the solution to be dried in a concentration ofat least or more than 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 8.0, 9.0 or 9.5%(w/v) and/or the polyol(s) are present in the solution to be dried in aconcentration of no more or less than 20.0, 17.5, 15.0, 12.5, 11.0 or10.5% (w/v). Most preferably, in this embodiment, the polyol(s) arepresent in the solution to be dried in a concentration of about 10%(w/v). The concentrations in this embodiment are e.g. suitable whensorbitol is used as polyol.

In another embodiment the polyol(s) are preferably present in thesolution to be dried in a concentration in the range of 0.1-40% (w/v),i.e. percent weight per volume of the solution. Thus, preferably, thepolyol(s) are present in the solution to be dried in a concentration ofat least or more than 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 12.5, 15.018.0, 19.0 or 19.5% (w/v) and/or the polyol(s) are present in thesolution to be dried in a concentration of no more or less than 50.0,40.0, 35.0, 30.0, 25.0, 22.5, 21.0 or 20.5% (w/v). Most preferably, inthis embodiment, the polyol(s) are present in the solution to be driedin a concentration of about 40% (w/v). The concentrations in thisembodiment are e.g. suitable when two different polyols are used such ase.g. sorbitol and mannitol. In that case the weight ratio between thetwo polyols may range from 1:100 to 1:1 including e.g. 1:50, 1:20, 1:10,1:5, and 1:2.

The Metal Salt

The metal salt dissolved in the solution to be dried can be anypharmaceutically acceptable salt of a divalent or monovalent metalcation. Preferred divalent cations are Ca⁺⁺, Mg⁺⁺ and Zn⁺⁺, of whichCa⁺⁺ and Mg⁺⁺ are more preferred, and of which Mg⁺⁺ is most preferred.Preferred monovalent cations are Li⁺, Na⁺ and K⁺, of which Li⁺, Na⁺ aremore preferred, and of which Li⁺ is most preferred. The counter-anion inthe metal salt preferably is not an amino acid, preferably not glutamateor aspartate. More preferably, the counter-anion in the metal salt aninorganic anion. Preferred (inorganic) anionic counterions are Cl⁻, SO₄²⁻ and CO₃ ²⁻, of which Cl⁻ and SO₄ ²⁻ are more preferred, and of whichCl⁻ is most preferred. The solution can comprise mixtures of one or moreof the above metal salts.

The metal salt(s) preferably are present in the solution to be dried ina concentration in the range of 0.005-10% (w/v), i.e. percent weight pervolume of the solution. Thus, preferably, the metal salt(s) are presentin the solution to be dried in a concentration of at least or more than0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0, 4.0, or 4.5%(w/v) and/or the metal salt(s) are present in the solution to be driedin a concentration of no more or less than 10.0, 8.0, 7.0, 6.0, or 5.5%(w/v). Most preferably the metal salt(s) are present in the solution tobe dried in a concentration of about 5% (w/v).

It is understood that in case the amino acid in the solution to be driedis dissolved in the form of a metal salt of the amino acid, e.g. MSG,the amino acid metal salt is not included in the weight percentage ofthe metal salt(s) but in the weight percentage of the amino acid(s),whereby the weight of the metal counter cation is included in the weightof the amino acid.

The Buffer

The solution to be dried preferably has a neutral pH, e.g. a pH in therange of 6.0-8.0, or 6.5-7.5 or a pH of about 7.0. The solution to bedried preferably comprises a buffer to maintain the pH at the indicatedvalues. In principle any pharmaceutically acceptable buffer, whichpreferably has effective buffering capacity in the range of pH values asindicated, can be used in the solution to be dried. The buffer ispreferably present in a concentration in the range of 0.5-100 mM, morepreferably in the range of 1-50 mM and most preferably in the range of2-20 mM, such as e.g. about 10 mM.

Suitable buffers for use in the solution to be dried include McIlvainebuffer (see Examples), a citrate buffer, a phosphate buffer, a HEPESbuffer and a histidine buffer.

Water

The water used for preparing the solution to be dried preferably isultrapure and preferably pyrogen-free water, like water for injections.

The concentrations of ingredients (e.g. the excipients) in the solutionto be dried of the invention are generally expressed herein as percentweight per volume (w/v) of the solution. This is understood to be therelationship of a solute (e.g. the excipient) to the solvent (e.g.water) expressed as grams of solute per liter of the total solution. Forexample 50 g of glucose in 1 L of solution, is considered a 5% w/vsolution.

In a preferred embodiment, the solution to be dried consists essentiallyof the biopharmaceutical agent in an amount as defined herein above,5-20% (w/v) sorbitol, 5-20% (w/v) monosodium glutamate, 2-10% (w/v) of amagnesium salt, preferably MgCl₂ and/or MgSO₄, optionally 5-20% (w/v)mannitol, and optionally 2-50 mM of a pharmaceutically acceptable bufferwhich buffers the solution at a neutral pH as indicated herein.

Drying

In the method of the invention, the aqueous solution comprising thebiopharmaceutical agent is dried. Any known drying method can be used. Adrying method can e.g. be spray drying, air drying, coating,foam-drying, desiccation, vacuum drying, vacuum/freeze drying orfreeze-drying, all of which are known to the person skilled in the artper se. In a preferred embodiment, the drying method is vacuum drying orfreeze-drying, of which freeze-drying or lyophilization is morepreferred.

In one embodiment of a freeze-drying process, the solution to be driedpreferably is first frozen to an initial freeze-drying or shelftemperature equal to or lower than of −50° C., −40° C., −30° C., −20° C.or −10° C. A preferred initial shelf temperature is equal to or lowerthan of −50° C. or −40° C. The solution to be dried may be subjected tofast freezing by immediately placing (a container/vial comprising) thesolution on the shelf having an initial shelf temperature as indicatedabove. Alternatively, the solution to be dried may be subjected to slowfreezing by placing (a container/vial comprising) the solution on theshelf having a temperature above 0° C., e.g. 2, 4 or 6° C., and thenslowly freezing the solution to the initial freeze-drying temperature asindicated above, by reducing the temperature, preferably at a rate ofabout 0.5, 1 or 2° C. per minutes. The solution to be dried may bebrought to a pressure of 100 microbar or lower. When the set pressurehas been reached, the shelf temperature may be increased to highertemperatures. The shelf temperature may e.g. be increased at a rate ofe.g. 0.05, 0.1 or 0.2° C. per minute to a temperature of 5, 10 or 15 or° C. above the initial freeze-drying temperature. The primary dryingstep is preferably ended when no pressure rise is measured in thechamber. Preferably at that moment, the shelf temperature may beincreased to e.g. 5, 10, 15, 20 or 25° C. at a rate of e.g. 0.01, 0.02or 0.05° C. per minute and optionally in one or more steps. During thesecondary drying phase the temperature is preferably kept at this valuetill no pressure rise can be detected. A preferred freeze-drying processis described in the Examples herein.

In one embodiment of a vacuum-drying process, the solution to be driedpreferably is at a temperature in the range of about 5-25° C., e.g. roomtemperature or more preferably at a temperature in the range of about10-20° C., e.g. a temperature of about 15° C. The pressure is thenreduced, e.g. to a pressure of less than 1, 0.5, 0.2, 0.1, 0.05 mbar.Once under reduced pressure the temperature of the solution being driedcan be decreased to a temperature that can be below 0° C. but that is(just) above the eutectic temperature of the solution to preventfreezing of the solution. When no pressure rise is measured in thechamber, the temperature of the solution can be increased to e.g. 5, 10,15, 20 or 25° C. at a rate of e.g. 0.01, 0.02 or 0.05° C. per minute andoptionally in one or more steps. The temperature is preferably kept atthis value till no pressure rise can be detected. A preferredvacuum-drying process is described in the Examples herein.

In a second aspect, the invention relates to a dry or dried formulationof a biopharmaceutical agent obtainable or obtained in a methodaccording to the invention as described herein above.

The methods of the invention for producing a dried formulation of abiopharmaceutical agent are preferably aimed at minimizing the loss ofactivity of the biopharmaceutical agent upon drying of the formulation.Preferably the methods of the invention as well as the formulationsthemselves are also aimed at minimizing the loss of activity of thebiopharmaceutical agent upon subsequent storage of the dried formulationobtainable with the methods of the invention. The methods of theinvention are thus preferably methods for producing stable formulationsof biopharmaceutical agents, i.e. formulations with a long or extendedshelf-life, preferably under refrigerated conditions (e.g. 2-10° C.), atroom temperature (e.g. 18-25° C.), or even at elevated temperatures(e.g. 32-45° C.) as may occur in tropical regions.

Loss of activity or inactivation of a biopharmaceutical agent isunderstood to include both loss of activity due to chemical pathways(such as oxidation, hydrolysis or enzymatic action) as well as physicalpathways (such as denaturation, aggregation, gelation). Preferably theloss of activity of the biopharmaceutical agent does not exceed anacceptable level. In other words, at least a level of biologicalactivity or viability and/or a level of original function or structuresufficient for the intended commercial therapeutic and/or diagnosticapplication of the biopharmaceutical agent is retained after dryingand/or subsequent storage.

Depending on the identity of the biopharmaceutical agent, the skilledperson will know which assay is to be used for assessing an activity ofsaid biopharmaceutical agent. The activity of the biopharmaceuticalagent may be expressed as its viability, e.g. in the case of active liveviruses, or activity may be expressed as enzymatic activity orbiological activity which may be determined in suitable assays known tothe skilled person. In other instances the activity of the may ratherrelate to the physical and chemical integrity of the agent and may bedetermined by assessing the structure and/or function of saidbiopharmaceutical agent. Antigen structure is preferably assessed byELISA or Biacor analysis. Secondary and tertiary structure is preferablyassessed by UV-, fluorescence, Fourier Transformed Infra Red (FTIR)and/or Circular Dichroism (CD) spectroscopy. Immunogenicity ispreferably assessed by in vivo analysis using animal models (e.g. mice,rats, cotton rats, ferrets or rabbits). Rats are e.g. a preferred modelfor poliovirus and cotton rats are a preferred model for RSV.

In a preferred embodiment of the methods of the invention, the drying ofthe solution (to be dried) causes the dried formulation upon rehydration(preferably immediately) after drying, to retain at least 50, 60, 70,75, 80, 85, 90 or 95% of the biological activity present in the solutionprior to drying. In case more than one biopharmaceutical agent ispresent in the formulation, the difference in loss of activities for thedifferent agents preferably is less than 50, 60, 70, 75, 80, 85, 90 or95%, whereby the retained activity of the agent with the most loss inactivity is expressed as percent of the retained activity of the agentwith the least loss, which is set at 100%.

In another preferred embodiment of the invention, the dried formulationretains at least 50, 60, 70, 75, 80, 85, 90 or 95% of the biologicalactivity present in the solution prior to drying, after storage for atleast one week at 45° C. and upon rehydration of the dried formulation.In case more than one biopharmaceutical agent is present in theformulation, the difference in loss of activities for the differentagents preferably is less than 50, 60, 70, 75, 80, 85, 90 or 95%,whereby the retained activity of the agent with the most loss inactivity is expressed as percent of the retained activity of the agentwith the least loss, which is set at 100%. Preferably the percentages ofactivities retained after storage as indicated above are also retainedafter storage two weeks, three weeks, four weeks, five weeks, six weeks,seven weeks, eight weeks, nine weeks, ten weeks or more at 37° C. or at45° C.

In a third aspect the invention relates to a formulation of abiopharmaceutical agent obtainable or obtained in a method according tothe invention as described herein above for use as a medicament. For useas a medicament the formulation can be used as dried formulation or itcan be reconstituted by dissolving the dried formulation, e.g. usingwater as defined above. The formulation is preferably reconstituted toits original volume, i.e. the volume before drying. Preferably theformulation is a formulation for inducing an immune response (in anindividual) against an infectious disease or a tumour. It is understoodthat the individual or subject to whom the formulations of the inventionare administered can be a human but can also be an animal, such as afarm animal or pet, including e.g. mammals, birds, livestock, poultry,cattle, bovines. More preferably, the formulation is a formulation forvaccination against an infectious disease or tumour. The formulation isthus preferably a formulation for the prevention or treatment of aninfectious disease or tumour. In another embodiment the inventionrelates to the use of the formulation obtainable or obtained in a methodaccording to the invention as described herein above for the manufactureof a medicament for inducing the immune response, for vaccination and/orfor the prevention or treatment of an infectious disease or tumour. Inyet another embodiment, the invention relates to a method for inducingan immune response against an infectious agent or tumour byadministering an effective amount of the formulation to a subject inneed thereof. The immune response is preferably induced against anantigen in the biopharmaceutical agent. The antigen preferably is anantigen of a pathogen causing the infectious disease or an antigen ofthe tumour, or an antigen that induces an immune response against thepathogen or the tumour. The pathogen preferably is a virus as hereindefined above. The formulation of the invention can be administered withor without reconstitution via intranasal, parenteral, intramuscular,subcutaneous and/or transdermal routes.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

DESCRIPTION OF THE FIGURES

FIG. 1 D-Antigen recovery directly after lyophilization of serotype 1, 2and 3 IPV-formulations (experiment A). The stabilizing effect of sucrose(SUC), trehalose (TREH), mannitol (MAN), dextran (DEX) and sodiumchloride (NaCl) was tested.

FIG. 2 Response surface plots representing D-antigen recoverypercentages of the three serotypes directly after lyophilization ofIPV-formulations containing 10% of mannitol and 0% dextran.

FIG. 3 D-Antigen recovery directly after lyophilization of serotype 1, 2and 3 IPV-formulations (experiment B). The stabilizing effect of sucrose(SUC), trehalose (TREH), mono-sodium glutamate (MSG), hydroxyethylstarch (HES) and sodium chloride was tested.

FIG. 4 D-Antigen recovery directly after vacuum drying (V),lyophilization with a slow freezing rate (L) and lyophilization with afast freezing rate (S). The stabilizing effect of sucrose and trehalose,compared to IPV vaccine without addition of stabilizers (negativecontrol) was investigated.

FIG. 5 D-antigen recovery directly after lyophilization of serotype 1, 2and 3 IPV-formulations as indicated (experiment C).

FIG. 6 D-antigen recovery directly after lyophilization of serotype 1, 2and 3 IPV-formulations (experiment D). All formulations contain 5%sorbitol and 125 mM NaCl and, excluding D11-D16, 5% peptone and arefurther as indicated.

FIG. 7 Accelerated stability testing of lyophilized serotype 1, 2 and 3IPV-formulations as indicated (experiment D) as determined by D-antigenrecovery after one week incubation at 45° C.

FIG. 8 D-antigen recovery directly after lyophilization of serotype 1, 2and 3 IPV-formulations as indicated (experiment E). The stabilizingeffects of sorbitol (SOR), sucrose (SUC), mannitol (MAN), mono-sodiumglutamate (MSG), peptone (PEP) and MgCl₂ were tested by using fast(panel A) and slow (panel B) freezing rates.

FIG. 9 Stability of lyophilized serotype 1, 2 and 3 IPV-formulations asindicated (experiment E) after one week incubation at 45° C. and usingfast (panel A) and slow (panel B) freezing rates. Only the mostpromising formulations directly after lyophilization are shown.

FIG. 10 D-antigen recovery directly after lyophilization of serotype 1,2 and 3 IPV-formulations (experiment F, n=3). The stabilizing effect ofpeptone was investigated in an IPV-formulation containing 10% sorbitol,10% MSG and 5% MgCl₂ with or without 10% mannitol. All formulations werefast frozen prior to the drying phase of the lyophilization process.

FIG. 11 Stability of lyophilized serotype 1, 2 and 3 IPV-formulations(experiment F, n=3) after one week incubation at 45° C. This experimentinvestigates whether it is possible to improve the IPV-formulationcontaining 10% sorbitol, 10% MSG and 5% MgCl₂ with or without 10%mannitol during accelerated stability with the addition of peptone orsingle amino acids.

FIG. 12 D-antigen recovery directly after lyophilization of serotype 1,2 and 3 IPV-formulations with different buffer components as indicated(experiment G). Four formulations as indicated in panels A, B, C and D,all containing 10% sorbitol, 10% MSG and were tested with 10 mMMcIlvaine, 10 mM citrate, 10 mM histidine, 10 mM HEPES and 10 mMphosphate buffer. The control formulation was not dialyzed.

FIG. 13 Stability of lyophilized serotype 1, 2 and 3 IPV-formulationswith different buffer components as indicated (experiment G) afterlyophilization and subsequent storage at 45° C. for one week. Fourformulations, containing 10% sorbitol, 10% MSG and MgCl₂ without or withmannitol and without or with peptone were tested in combination with a10 mM McIlvaine, 10 mM citrate, 10 mM histidine, 10 mM HEPES and 10 mMphosphate buffers. The control formulations were not dialyzed and 1:1diluted with McIlvaine buffer containing the excipients as indicated.

FIG. 14 D-Antigen recovery directly after vacuum drying (V),lyophilization with a slow freezing rate (L) and lyophilization with afast freezing rate (S). The stabilizing effect of different formulationscompared to IPV vaccine without addition of stabilizers (negativecontrol) and sucrose or trehalose formulated IPV.

FIG. 15 D-antigen recovery directly after lyophilization of serotype 1,2 and 3 IPV-formulations as indicated.

FIG. 16 Stability of serotype 1, 2 and 3 IPV-formulations as indicatedafter lyophilization and subsequent storage at 45° C. for one week, asdetermined by D-Antigen recovery.

EXAMPLES

1. Materials and Methods

1.1 Materials

The trivalent inactivated polio vaccine (Salk-IPV), containing theinactivated Mahoney strain for type 1, MEF for type 2 and Saukett fortype 3, was obtained from the process development department of theRIVM-Vaccinology (Bilthoven, The Netherlands). The Salk-IPV trivalentbulk (10×) was formulated as a ten times concentrated 40-8-32 DU/singlehuman dose (1 ml). The concentration of the IPV 05-126B bulk that wasused in this study was determined at 411-90-314 DU/ml with the QC-ELISAas described by Westdijk et al. [6].

The excipients sucrose, D-sorbitol, D-trehalose dihydrate, D-glucosemonohydrate, mannitol, L-glutamic monosodium salt monohydrate (referredto as glutamate, sodium glutamate, monosodium glutamate or MSG herein),myo-inositol, D-raffinose, hydroxy ethyl starch, glycine, L-proline,L-leucine, calciumchloride dihydrate, maltitol, magnesiumchloridehexahydrate, lithium chloride, and ovalbumin were all purchased fromSigma (St. Louis, Mo.). Peptone (vegetable), dextran (6 kDa, fromLeuconostoc ssp), L-histidine, L-alanine, zinc chloride, calciumlactobionate monohydrate were from Fluka (Buchs, Switzerland). Lactitol(Lacty®-M) was from Purac Biochem (Gorinchem, The Netherlands),L-arginine (EP, non-animal origin) and Tween80 were from Merck(Darmstadt, Germany), polyvinylpyrrolidone 25 (PVP, 29 kDa) was fromServa Feinbiochemica GmbH (Heidelberg, Germany), Sol-U-Pro, a hydrolyzedporcine gelatin, was from Dynagel Inc. (Calumet City, Ill.) and Ficollwas from Pharmacia (Uppsala, Sweden). As buffer components sodiumdihydrogen phosphate dihydrate (NaH₂PO₄), sodium chloride (NaCl),potassium dihydrogen phosphate (KH₂PO₄) and EDTA from Merck were used.Trisodium citrate dihydrate, citric acid and HEPES were fromSigma-Aldrich (St. Louis, Mo.) and disodium hydrogen phosphate dihydrate(Na₂HPO₄) was from Fluka (Buchs, Switzerland). All excipients used wereof reagent quality or higher grade.

To prepare 10 mM McIlvaine buffer, 10 mM citric acid was added to 10 mMNa₂HPO₄ in a ratio of 1:6 and a pH-value of 7.0. For the 10 mM citratebuffer the components trisodiumcitrate dihydrate (10 mM) and citric acid(10 mM) were mixed together till pH 7.0 was reached. The 10 mM phosphatebuffer of pH 7.0 consisted of 10 mM KH₂PO₄ and 10 mM Na₂HPO₄. The 10 mMHEPES and 10 mM histidine buffers were prepared by weighing anddissolving the buffer components followed by adjustment of the pH-valueat 7.0 using HCl and/or NaOH.

1.2 Methods

1.2.1 Dialysis

Unless otherwise indicated, the trivalent IPV bulk material was dialyzedagainst 10 mM McIlvaine buffer (pH 7.0) using a 10 kDa molecular weightcut-off, low-binding regenerated cellulose membrane dialysis cassette(Slide-A-Lyzer®, Pierce, Thermo Scientific, Rockford, Ill.) to replacethe buffer components of the IPV bulk (M199 medium).

1.2.2 Solutions to be Dried

All excipients were dissolved in McIlvaine buffer at a doubleconcentration of the indicated end concentration. The dialyzed IPV wasequally mixed with the formulation to be tested. Subsequently 2 ml glassinjection vials (Müller+Müller, Holzminden, Germany) were filled with0.2 ml of the IPV-excipient mixtures and provided with 13 mm pre-dried(overnight at 90° C.) rubber stoppers (type V9250 from Helvoet Pharma,Alken, Belgium).

1.2.3 Lyophilization and Vacuum-Drying Process

For lyophilization, filled and half-stopped vials were loaded into aLeybold GT4 freeze-dryer or Zirbus pilot/laboratory freeze-drying unitsublimator 2-3-3 at a shelf temperature of −50° C., or at a shelf of 4°C. and then frozen to −50° C. by reducing the temperature at a rate of1° C./min, which will be denoted as fast and slow freezing,respectively. The vials were kept at a temperature of −50° C. for 2 h.For the primary drying phase the shelf temperature was increased at 0.1°C./min to −45° C., then at 0.02° C./min to −40° C., followed byincubation for 42 h. The secondary drying phase was performed by furtherincrease of the shelf temperature at 0.02° C./min to 10° C., followed byan 8 h during incubation at 10° C. Thereafter, the shelf temperature wasincreased at 0.02° C./min to 25° C.

For vacuum drying, filled and half-stopped vials were loaded into aZirbus freeze-drying unit sublimator 2-3-3 at a shelve temperature of15° C. and kept at that temperature for 10 minutes. The chamber pressurewas reduced till 1 mbar in ramping steps of 15 minutes with differentrates (1 mbar/min, 0.3 mbar/min, 0.1 mbar/min) and starting at a 25 mbarchamber pressure. The temperature was decreased till −10° C. for 1 h at0.05 mbar and for 1 h at 0.03 mbar, resulting in no freezing of theformulations (product temperature above eutectic temperature of theformulations). Subsequently, shelf temperature was increased at 0.05°C./min to 30° C. At the end of the cycle, the vials were closed undervacuum, sealed with alu-caps and kept at 4° C. until analysis. Anexample of the shelf temperatures and chamber pressures during thecourse of vacuum drying process is shown in Table 1.

TABLE 1 T_(shelf) Period Pressure (° C.) (min) (mbar) FT01 15 10 — D0115 15 25 D02 15 15 10 D03 15 15 5 D04 15 15 3 D05 15 15 1 D06 −10 600.05 D07 −10 60 0.03 D08 −5 120 0.03 D09 5 120 0.03 D10 10 120 0.03 P0120 240 — P02 30 240 — P03 4 60 —1.2.4 D-Antigen ELISA

Polystyrene 96-well microtiter plates were coated overnight at roomtemperature with serotype-specific bovine anti-polio serum (RIVM,Bilthoven, The Netherlands). After washing with 0.1% Tween20 in PBS(wash buffer), twofold dilutions of an IPV reference standard and asingle dilution of IPV-formulations diluted in assay buffer (PBS with0.5% Protifar and 0.1% Tween20) were added (100 μl/well, in duplicate).The plates were incubated at 37° C. for 30 minutes under gentle shaking,extensively washed and a mixture of serotype-specific monoclonal mouseantibodies (mab 3-4-E4 (type 1), 3-14-4 (type 2), 1-12-9 (type 3), allfrom RIVM, Bilthoven, The Netherlands) and HRP-labeled anti-mouse IgG(GE Healthcare, Buckinghamshire, UK) was added. Subsequently, plateswere incubated at 37° C. for 30 minutes under gentle shaking. Plateswere washed extensively and ELISA HighLight signal reagent from(Zomerbloemen BV, Zeist, The Netherlands) was added andchemiluminescence was measured during 10-15 minutes by using aluminometer (Berthold Centro LB960).

1.2.5 Moisture-Content Analysis

The water content was determined using a Karl Fischer coulometrictitrimeter (Model CA-06 Moisture meter, Mitsubishi). The principle ofthe water residue determination by Karl Fischer method is based on thefact that iodine and sulphurdioxide only react in the presence of water.The samples were weighted and subsequently reconstituted in theKarl-Fischer reagent, Hydranal Coulomat A (Fluka, Buchs, Switzerland).The reconstituted sample was withdrawn into a syringe and injected intothe titration vessel. Each vial was measured in triplicate. The emptyvials were weighted and the water content was calculated based on thewater content measured by the titrimeter, the weight of the lyophilizedproduct in the vial, the reconstitution volume of the reagent, titrationvolume and the water content of the blank titration.

1.2.6 Differential Scanning Calorimetry (DSC)

The thermodynamic behaviour of the formulations was determined bydifferential Scanning calorimetry (DSC), a method which measures thetemperatures and heat flow, associated with phase transitions inmaterials, as a function of time and temperature. The freeze-driedformulations were filled in an aluminium DSC pan and subjected to acontrolled temperature program in a differential scanning calorimeter(DSC Q100, TA Instruments). The sample was heated from 0° C. to 150° C.at a heating rate of 20° C./min and the sample chamber was purged withnitrogen gas (50 ml/min). The glass transition temperatures (Tg) weredetermined as the midpoint of the discontinuities in the heat flowcurves using software (Universal Analysis 2000, TA Instruments).

2. Results

2.1 Stabilizing Different IPV Subtypes During Lyophilization

In the first experiment (Experiment A) four well known stabilizingsugars/polyols (sucrose, trehalose, mannitol and dextran), as well assodium chloride were evaluated for their stabilizing potential (FIG. 1).The study was set up with a design of experiments approach in order toobtain an optimal formulation for lyophilization of IPV.

Different IPV-formulations were lyophilized as described above (section1.2.3). Lyophilized cakes were reconstituted by adding an equal amountof water as the starting volume and the D-antigen recovery wasdetermined by an ELISA (section 1.2.4). Recoveries were shown as thepercentages of the D-antigen content in the liquid formulations, whichwere measured before lyophilization.

The trivalent IPV formulation, without any additives, IPV 1:1 dilutedwith McIlvaine buffer, showed recoveries <10% for all serotypes afterlyophilization (FIG. 1; A1). Type 2 IPV showed to be the most stableserotype in all formulations with maximum D-antigen recoveries of ±80%.Dextran seemed to have a negative effect on D-antigen recovery of thelyophilized IPV formulations, especially for type 1 and 3. Best results,with maximum recoveries of ±55%, ±85% and ±50% for serotype 1, 2 and 3respectively, were obtained with formulations containing sucrose and/ortrehalose in combination with mannitol (FIG. 1; formulations A3, A4, A12and A16). Addition of NaCl had no positive effect on the recovery of IPVafter lyophilization.

This first pilot experiment clearly shows the complexity of lyophilizinga trivalent polio vaccine in which each IPV serotype prefers its ownstabilizing agents. In a formulation with 10% mannitol type 1 and type 3preferred the presence of high concentrations sucrose without trehalose,whereas type 2 preferred a high concentration of trehalose withoutsucrose (FIG. 2).

In the next experiment (Experiment B) the stabilizing potential of amixture of glutamate, a saccharide, and a polymer was investigated.Different combinations of the excipients sucrose, trehalose, monosodiumglutamate (MSG), hydroxyethyl starch (HES) and NaCl were investigated.Lyophilization of trivalent IPV with formulations based on MSG togetherwith disaccharide, sucrose and/or trehalose, showed D-antigen recoveriesof 50-60%, 70-95% and 50-65% for the three serotypes respectively (FIG.3; formulations B4, B7, B9, B12, B13). Formulations based on only 8% HESor 63 mM NaCl did not protect IPV during lyophilization (FIG. 3; B5 andB10). The addition of NaCl to a formulation with sucrose showed an 5-10%increase in D-antigen recovery after lyophilization (FIG. 3; B2 andB16).

2.2 Impact of the Drying Process and Formulation

In the next test results are shown of typical formulations used fordrying of biopharmaceuticals in relation to the drying process.Formulations containing trivalent IPV were dried by vacuum drying (adrying method without freezing), freeze drying using a fast freezingstep (direct placement of the product on pre-cooled shelves of −50° C.)and freeze drying using a slow freezing step (placement of product onshelves of 4° C. and freezing towards −50° C.). As shown in FIG. 4standard formulations, e.g. based on sucrose or trehalose, partiallyprotect IPV upon vacuum drying. However, these formulations do not giveprotection upon freeze drying.

2.3 Screening of Excipients

In the next experiment (Experiment C) different formulations containingsorbitol, mannitol, sucrose and/or MSG combined with some amino acids,proteins/peptides or other stabilizing agents were tested (Table 2). Inorder to investigate the effect of salt in the lyophilizedIPV-formulation, the C-formulations were also tested with addition of125 mM NaCl. No clear effect of the NaCl on the D-antigen recoveries wasobserved (data not shown). Having a first look on the antigenicityresults directly after lyophilization, it was clear that formulationC13, containing 5% sorbitol, 5% peptone and 1% lithium chloride (LiCl),showed the highest recoveries for all serotypes; ±85%, ±100% and ±85%for type 1, 2 and 3 respectively (FIG. 5). Substitution of theLiCl-compound in 1.8% MgCl₂ resulted in a decrease of ±5%±20% and 15% inD-antigen recovery for the three serotypes. However, these formulationsshowed relative high residual moisture contents, 2.1% for theMgCl₂-containing formulation and 6.2% for the formulation with LiCl. Theaddition of a very small amount of surfactant Tween 80 to a formulationcontaining sucrose, trehalose and the amino acids glycine and lysineincreased the D-antigen recoveries with 5-15% (FIG. 5; C8, C9). Theformulations based on sorbitol, mannitol and MSG (C2, C3, C4 and C7)showed recoveries of more than 65%, 80% and 70% for type 1, 2 and 3respectively.

In this study, the combination of sorbitol, peptone and the salts LiClor MgCl₂ seemed to have a positive effect on the D-antigen recoverydirectly after lyophilization of IPV. Another notable formulation is themixture of sorbitol, mannitol and MSG, which showed that the presence ofpolyols in combination with MSG stabilizes the IPV duringlyophilization.

The glass transition temperature of the lyophilized formulations wasmeasured (Table 2), but showed no clear relation with the D-antigenrecoveries. Formulations containing ovalbumine and peptone showed thehighest glass transition temperatures.

TABLE 2 Composition of the lyophilized IPV-formulations (Experiment C).Residual moisture content (RMC) was determined by Karl Fischer and theT_(g) of the dried cake by DSC. Amino RMC T_(g) SOR MAN SUC MSGSugars/polyols acids Proteins Other (%) (° C.) C1 — — — — — — — — 3.6n.d. C2 7% 7% — 2% — 2% 7% — 0.3 37.2 Glycine Ovalbumin C3 7% 7% — 2% —2% 7% 1 mM n.d. 38.3 Glycine Ovalbumin EDTA C4 7% 7% — 2% — 2% — — 1.953.5 Glycine C5 — — 3% — 3% Dextran — 3% — 1.1 54.9 3% Myo- OvalbuminInositol C6 5% 5% 5% — — 2% — — 1.0 37.1 Glycine 3% Lysine 3% L- Arg C75% 5% 5% 3% — 2% — — 3.3 37.2 Glycine 3% Lysine C8 — — 5% — 5% Trehalose3% — — 0.5 32.3 Lysine 3% Alanine C9 — — 5% — 5% Trehalose 3% — 0.01%0.5 34.1 Lysine Tween80 3% Alanine C10 — — 5% — — 3% — 3% Ca- 1.0 31.2Lysine Lactobionate 3% Alanine C11 — — 5% — — 3% 3% Rec. — 0.3 35.7Lysine Gelatin 3% Alanine C12 5% — — — — — 5% 1.8% MgCl₂ 2.1 44.7Peptone C13 5% — — — — — 5% 1% LiCl 6.2 n.d. Peptone C14 — — 5% — 5%Trehalose — 5% — 0.5 35.6 Peptone

Based on these findings a new screening experiment (Experiment D) wasdesigned. Since the most promising recoveries were obtained withformulations based on sorbitol, peptone and Mg or Li-chloride, wedesigned an experiment based on 10% sorbitol, 5% peptone and 125 mMNaCl. Ovalbumine was discarded since it is from animal origin, thus anundesirable excipient in a vaccine for human use. In order to get moreinsight in the IPV stabilizing mechanism of several excipients,formulations containing 10% sorbitol, 5% peptone and 125 mM NaCl werecombined with either a sugar/polyol, an amino acid (instead of 5%peptone), a salt or other stabilizing agents, like surfactants orproteins. The formulation with sorbitol, NaCl and 1% histidine showedrecoveries of 90-100% directly after the freeze-drying process (FIG. 6;D11). As observed earlier, the MgCl₂-containing formulation showedauspicious stabilizing capacity during lyophilization, which was similarfor the calciumchloride (CaCl₂)- and lithium chloride (LiCl)-containingIPV formulations (FIG. 6; D17-19). During the screening phaseaccelerated stability has been evaluated to select the excipients ontheir ability to provide a stable lyophilized product as well, evenafter subsequent storage at high temperature.

2.4 Accelerated Stability Testing

Although some formulations showed acceptable D-antigen recoveriesdirectly after lyophilization, after one week incubation at 45° C. theD-antigen recoveries of these four formulations were dropped tillpercentages below 30%, 60% and 10% for respectively serotype 1, 2 and 3(FIG. 7). The formulation with 5% myo-inositol represented the highestrecoveries after incubation at 45° C., but still a decrease of ±40%, 10%and 30% was observed for the three serotypes, which showed again IPVtype 2 to be the most stable serotype (FIG. 7; D5). Upon one weekstorage at 45° C., the formulation containing 1% lactitol showed lessthan 10% decrease in D-antigen recovery for serotype 2, unfortunatelyserotype 1 and 3 showed a decrease of >30% (FIG. 7; D7).

In order to further investigate the combination with sorbitol, mannitol,MSG and the stabilizing potential of peptone and MgCl₂, a new design ofexperimental set up was performed to determine the relationship betweenthe different excipients and D-antigen recovery after lyophilization.The following variables were included in Experiment E: 0/10% sorbitol,0/10% sucrose, 0/10% mannitol, 0/10% MSG, 0/5% peptone and/or 0/5% MgCl₂and freezing speed was investigated in this experiment. Slow freezingmeans that the vials were placed on shelves at 4° C. and subsequentlycooled till −50° C. at a rate of 0.1° C./min, where fast freezing meansthat the vials were directly placed at shelves pre-cooled at −50° C. Theresults are shown in FIG. 8A for the fast freezing rate and in FIG. 8Bfor the slow freezing rate.

Having a first look on the D-antigen recoveries after lyophilization,the fast frozen formulations containing MSG and MgCl₂ in combinationwith a sugar/polyol showed the highest recoveries of ±80-90% for allserotypes (FIG. 8A; E22-24, E29-32). For the slow frozen samples theMgCl₂-containing formulations showed recoveries of 75-90% for type 1 and3 (FIG. 8B; E54-57, E63-66). However, for serotype 2 only formulationsof the sugar(s) in combination with peptone showed recoveries of ±70%(FIG. 8B, E43-48). To have an indication of the reproducibility of theexperiment formulation H33 and H66 were tested in triplicate and showedstandard deviations <10% for all serotypes. From the comparison of theD-antigen recoveries of these two formulations, directly afterlyophilization (FIG. 8) or after one week incubation at 45° C. (FIG. 9),it is clear that freezing rate did not influence the recoverysignificantly for these IPV formulations.

Experiment E was set up on the basis of ‘Design of Experiment’ using the“Modde” software from Umetrics. Besides recovery of D-antigen afterlyophilization, also recoveries after lyophilization and subsequentstorage at 37° C. or 45° C. were used as output in the design. Theoutput as function of the formulations was modulated and put in a modelusing Modde. This revealed which formulation parameters affected therecovery after lyophilization and storage (data not shown).

The most important formulation parameters for each of the viral subtypesare summarized in Tables 3-5.

TABLE 3 Recovery of D-antigen of type 1 formulation after after storageafter storage parameter lyophilization % at 37° C. % at 45° C. % MSG 910 7 Sorbitol 8 6 4 MgCl₂ 7 6 3 Peptone 6 4 Mannitol 4 6 MSG* MgCl₂ 4

TABLE 4 Recovery of D-antigen of type 2 formulation after after storageafter storage parameter lyophilization % at 37° C. % at 45° C. % MSG 7 99 Sorbitol 7 7 5 MgCl₂ Peptone 10 10 Mannitol 3 4 4 Sucrose 2

TABLE 5 Recovery of D-antigen of type 3 formulation after after storageafter storage parameter lyophilization % at 37° C. % at 45° C. % MSG 1410 10 Sorbitol 8 7 7 MgCl₂ 4 4 4 Peptone 4 12 12 Mannitol 6 5 5 Sucrose4 1 1 MSG* MgCl₂ 5 52.5 Substitution of Peptone

Since peptone seemed to stabilize the lyophilized IPV during thesubsequent storage at a temperature of 45° C., we performed anexperiment to investigate the role of peptone in a formulation with 10%sorbitol, 5% MSG and 5% MgCl₂ and the same formulation combined with 10%mannitol. No significant differences were found with the addition of 10%mannitol to the formulation containing sorbitol, MSG and MgCl₂. Adding5% peptone did not affected the antigenicity of both formulations (FIG.10).

To find out whether the addition of single amino acids could take overthe stabilizing role of peptone during subsequent storage of thelyophilized IPV, amino acids were added to the formulation containingsorbitol, MSG and MgCl₂ with or without mannitol. After a weekincubation at 45° C. neither peptone or one of the added amino acidsshowed improved stability of the D-antigen recovery when compared to thecontrol formulation, which contain 10% sorbitol, 10% MSG and 5% MgCl₂(FIG. 11, F1.0-F1.5). In the presence of mannitol the stabilizingcapacity of peptone was only shown for type 3, whereas the D-antigenrecovery was improved with ±15% when compared to the control sample.However, a significant decreased recovery (p<0.001) was found for type 1and, even though not significant, type 2 showed also a ±10% lowerrecovery (FIG. 11; F2.0, F2.1). For all formulations containingsorbitol, MSG and MgCl₂ a decrease of ±10% of D-antigen content type 2was shown after one week 45° C., where the addition of mannitol wasfound to be stable for type 2 during accelerated stability. Arginineseemed to have stabilizing potential in a formulation (FIG. 11, F2.3)with sorbitol, MSG, MgCl₂ and mannitol and showed only ±15% and ±30%decrease in D-antigen recovery for type 1 and 3 respectively afterincubation at 45° C. This was comparable to the stabilization by almostthe same formulation with peptone instead of arginine (FIG. 11, F2.1).

Due to the fact that an undefined excipient, such as peptone, is notpreferred in a human vaccine, a possible substitute for peptone, whichcould stabilize the IPV during storage, was investigated. Analysis bymass spectrometry and HPLC showed the most abundant amino acids presentin peptone (data not shown). The addition of several single amino acidsto the formulation containing sorbitol, MSG and MgCl₂ did not improvethe stability at 45° C. when compared to the control formulation. Wherepeptone seemed to stabilize serotype 3 in the formulation containingsorbitol, MSG, MgCl₂ and mannitol, arginine is able to improve thestability of both serotype 1 and 3. Serotype 2 showed already in thecontrol formulation full maintenance of D-antigen recovery duringaccelerated stability. Although the exact composition of peptone is hardto determine, the amino acid quantification by reverse-phase HPLC withnon-hydrolyzed versus chemical hydrolyzed peptone showed that peptoneconsists of both single amino acids and peptides, however >90% w/w ofthe peptone remains undefined. Since peptone seemed to increase glasstransition temperature of the studied IPV-formulations, it might bepossible to replace the peptone by an excipient with high T_(g), likesucrose or trehalose.

TABLE 7 Glass transition temperatures (Tg′ and Tg) of IPV- formulationscontaining sorbitol, MSG and MgCl₂ with/without mannitol were determinedby DSC. The effect of peptone on the glass transition of theseformulations was investigated. Single measurements were shown. 10%sorbitol + 10% MSG + 10% sorbitol + 10% MSG + 5% MgCl₂ 5% MgCl₂+ 10%mannitol T_(g)′ (° C.) T_(g) (° C.) T_(g)′ (° C.) T_(g) (° C.) Control−47.9 35.2 −44.4 38.8 +5% −44.1 39.8 −42.8 48.7 peptone

The previous experiment did not yielded a worthy substitute for peptoneand showed that the formulations with sorbitol, MSG, MgCl₂ with orwithout mannitol gave the best results, even after accelerated stabilitytests. During this study all formulations were prepared with McIlvainebuffer, which is known to be a suitable buffer for lyophilization of IPV[52]. In order to further optimize the formulation a buffer screeningwas performed with buffers that are frequently used for lyophilizationof biopharmaceuticals [18]. IPV batches in each buffer were prepared bydialysis and non-dialyzed IPV acted as control.

The formulation with sorbitol, MSG and MgCl₂ showed recoveries of ±95%,±85% and ±90% for the three serotypes directly after lyophilization(FIG. 12A; G1.0-G1.5). After addition of peptone the type 1 and 3D-antigen recoveries were dropped with 10-15%, where type 2 showedsimilar recoveries when compared to the formulation without peptone(FIG. 12B). McIlvaine buffer showed 10-15% lower recoveries for serotype2 in comparison with the other buffer components. Histidine bufferseemed to increase the D antigen yield of IPV after lyophilization theIPV-formulation with sorbitol, MSG and MgCl₂ (recoveries of >95% fortype 1 and 3; FIG. 12A, G1.3). After lyophilization, a D-antigenrecovery of 88% for type 2 was reached with the phosphate buffer, wherethe McIlvaine buffer showed 73% recovery for type 2 (FIG. 12A, G1.5).

The addition of mannitol to the formulations of sorbitol, MSG, MgCl₂.without peptone revealed that serotype 2 prefers the presence ofmannitol in the formulation during lyophilization, since recoveries of85-100% for type 2 were found (FIG. 12C). For type 1 recoveries of80-90% and for type 3±80% were found for the sorbitol, MSG, MgCl₂ andmannitol containing IPV-formulations. The addition of peptone showed±10% reduction in recovery of type 1 and 3 and similar D-antigencontents for type 2 after lyophilization (FIG. 12D).

2.6 The Effect of Buffer Components

An accelerated stability experiment showed formulation-dependentdifferences between the used buffers. Whereas the control formulationsshowed high recoveries directly after lyophilization, recoveries of±40%, ±50% and <15% for serotype 1, 2 and 3, respectively, were foundfor the peptone-lacking control formulations after one week incubationat 45° C. (FIG. 13; G1.0 and G3.0). For the control formulations,peptone showed clear improvement of stability of type 1 and 2 when wheretype 3 showed a decrease of ±25% and ±15% in D-antigen recovery, insteadof >90% and 80%, respectively for the formulations without and withmannitol after storage at 45° C. (FIG. 13; G2.0 and G4.0). For theformulation containing sorbitol, MSG and MgCl₂ the highest recoverieswere observed with HEPES buffer, D-antigen contents of ±80%, ±85% and±60% for the three serotypes after lyophilization and storage. Thismeans that only 10%, 0% and ±30% reduction in D-antigen recoveryoccurred during accelerated stability testing for type 1, 2 and 3respectively. Only for type 3 a better result was observed with citratebuffer; a reduction of ±20% and remained recovery of 79% (FIG. 13A;G1.2). Addition of peptone showed overall 15-25% lower recoveries forall serotype when compared to the same formulation and buffer withoutpeptone (FIG. 13A). Obvious improved stability was shown with theaddition of peptone to the formulation with sorbitol, MSG, MgCl₂ andmannitol. Where the different buffer formulations without peptone showedmaximum recoveries of ±55%, ±75% and ±45%, the same buffers with peptoneshowed recoveries of ±50%, ±80% and ±55% (FIG. 13B). The formulationbased on phosphate buffer with the excipients sorbitol, MSG, MgCl₂,mannitol and peptone showed to be the most stable formulation withreduced recoveries of ±30% for type 1 and only ±10% for type 2 and 3after incubation at 45° C.

2.7 Formulations Suitable for Stabilization Independent of Drying Method

FIG. 14 shows that only formulations containing sorbitol can resiststresses caused by the different drying methods. This could further beimproved by inclusion of MSG and MgCl₂ (especially type 3), resulting informulations that gave comparable or even higher recoveries than vacuumdrying with trehalose or sucrose. From earlier results (Experiment E) weconcluded that the formulations based on sorbitol, MSG and MgCl₂possesses the best recovery after accelerated stability testing.

2.8 Formulations without Peptone

One of the better formulation without peptone that we found in aboveexperiments, is a formulation containing 10% sorbitol+10% MSG+5% MgCl₂.In an additional experiment we evaluated what the impact was of theprimary excipients in this formulation. The results were compared with 2standard formulations based on sucrose and trehalose. The results directafter lyophilization are shown in the FIG. 15. From FIG. 15 we concludethat primarily sorbitol is responsible for recovery of IPV afterlyophilization, especially for subtypes 1 and 2. The combination of MSGand MgCl₂ shows significant stabilization of the three IPV subtypes.Compared to sorbitol only, the combination of sorbitol, MSG and MgCl₂,showed the best recoveries after lyophilization, especially for type 3.

Upon accelerated stability evaluation as shown in FIG. 16, most of theformulations showed an enormous decrease in recovery, also theformulation containing only sorbitol. However, the formulationscontaining 10% sorbitol+10% MSG+5% MgCl₂ (with/without mannitol) showedhigh recoveries after storage at 45° C. for one week.

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ABBREVIATIONS

BCG—Bacillus Calmette-Guérin

DoE—Design of Experiments

DSC—Differential Scanning calorimetry

DU/D-Ag—D-Unit/D-antigenicity

ELISA—Enzyme-Linked ImmunoSorbent Assay

HES—Hydroxyethyl starch

HRP—Horseradish peroxidase

IgG—Immunoglobulin G

IPV—Inactivated Polio Vaccine

MAN—Mannitol

MS—Mass Spectrometry

MSG—Mono-Sodium Glutamate

OPV—Oral Polio Vaccine

PEP—Peptone

QC—Quality Control (department at RIVM)

RIVM—National Institute for Public Health and Environment

RMC—Residual Moisture Content

RP-HPLC—Reversed-Phase High-Performance Liquid Chromatography

sIPV—Sabin Inactivated Polio Vaccine (based on Sabin strains)

SOR—Sorbitol

SUC—Sucrose

T_(g)—Glass transition temperature

TREH—Trehalose

VAPP—Vaccine Associated Paralytic Poliomyelitis

VDPV—Vaccine Derived Poliovirus

WHO—World Health Organisation

The invention claimed is:
 1. A method for producing a formulation of abiopharmaceutical agent, comprising drying a solution comprising: (a) abiopharmaceutical agent comprising poliovirus, (b) an amino acidselected from the group consisting of glutamate, arginine, histidine,glycine and mixtures thereof, (c) a polyol comprising sorbitol and/ormannitol, and (d) at least 0.2% (w/v) of a metal salt and water, whereinthe metal salt is Mg²⁺, Ca²⁺, Li⁺ or a mixture thereof.
 2. The methodaccording to claim 1, wherein the solution consists essentially of 1pg-10 g per ml of the biopharmaceutical agent, 0.01-20% (w/v) of theamino acid, 0.5-20% (w/v) of the polyol, 0.2-10% (w/v) of the metal saltand water.
 3. The method according to claim 1, wherein the glutamate isdissolved in the solution in the form of monosodium glutamate, and/orwherein the arginine is in the form of poly-L-arginine.
 4. The methodaccording to claim 3, wherein the solution consists essentially of 1pg-10 g per ml of the biopharmaceutical agent, 5-20% (w/v) sorbitol,5-20% (w/v) monosodium glutamate, 2-10% (w/v) of a magnesium salt, andoptionally 5-20% (w/v) mannitol.
 5. The method according to claim 1,wherein the solution comprises a pharmaceutically acceptable buffer andis buffered at a neutral pH.
 6. The method according to claim 1, whereinthe drying is by air drying, vacuum drying, spray drying or bylyophilization.
 7. The method according to claim 1, wherein thepoliovirus is one or more of poliovirus serotypes 1, 2 or
 3. 8. Themethod according to claim 1, wherein the poliovirus is inactivated. 9.The method according to claim 1, wherein the formulation, uponreconstitution in a liquid, retains at least 50% of the activity of thebiopharmaceutical agent present in the solution prior to drying.
 10. Themethod according to claim 9, wherein the formulation comprises at leasttwo different poliovirus serotypes, and wherein the difference in lossof activities for the different agents is less than 50%, whereby theretained activity of the agent with the most loss in activity isexpressed as percent of the retained activity of the agent with theleast loss, which is set at 100%.
 11. The method according to claim 1,wherein the formulation upon reconstitution after storage for at leastone week at 45° C., retains at least 50% of the activity of thebiopharmaceutical agent present in the solution prior to drying.
 12. Themethod according to claim 11, wherein the formulation comprises at leasttwo different poliovirus serotypes, and wherein the difference in lossof activities for the different agents is less than 50%, whereby theretained activity of the agent with the most loss in activity isexpressed as percent of the retained activity of the agent with theleast loss, which is set at 100%.
 13. The method according to claim 4,wherein the magnesium salt is MgCl₂ and/or MgSO₄.
 14. A method forproducing a formulation of a biopharmaceutical agent, comprising dryinga solution comprising poliovirus, glutamate, sorbitol, and at least 0.2%(w/v) of Mg²⁺ metal salt and water.
 15. The method according to claim14, wherein the drying is by lyophilisation.
 16. The method according toclaim 15, wherein the drying is by vacuum drying.